Glutathione‐Responsive Nanoparticles of Camptothecin Prodrug for Cancer Therapy

Abstract Camptothecin (CPT) is a potent chemotherapeutic agent for various cancers, but the broader application of CPT is still hindered by its poor bioavailability and systemic toxicity. Here, a prodrug that releases CPT in response to glutathione (GSH), which is commonly overexpressed by cancer cells is reported. Through assembling with PEGylated lipids, the prodrug is incorporated within as‐assembled nanoparticles, affording CPT with a prolonged half‐life in blood circulation, enhanced tumor targetingability, and improved therapeutic efficacy. Furthermore, such prodrug nanoparticles can also promote dendritic cell maturation and tumor infiltration of CD8+ T cells, providing a novel strategy to improve the therapeutic efficacy of CPT.


Contents
anti-mouse I-A/I-E Antibody, APC anti-mouse CD4 were purchased from BioLegend (USA).

Instrumentation and Methods
1 H-and 13 C-NMR spectra were recorded on a 400 MHz NMR spectrometer (Bruker).

Determination of the reaction kinetics of CPT
In vitro cellular uptake of S-NP-CPT by CLSM and flow cytometry: Then 10 mL of distilled water was added, which was further sonicated for 10 min.
Intracellular uptake of NP-Cy5.5 was studied by CLSM and flow cytometry. For CLSM observation, CT26 cells were seeded in 24-well chamber slides (Thermo Scientific, USA) at a density of 2 × 10 4 cells per well and incubated with RPMI1640 supplemented with 10% FBS (1 mL) at 37 °C for 12 h. After removing the medium, cells were treated with NP-Cy5.5 at an equivalent Cy5.5 concentration (10 μg/mL) for 0.5 h, 3 h, and 6 h, respectively. The medium was removed and cells were incubated with FITC phalloidin according to the manufacturer's protocol. Subsequently, the cells were stained with DAPI and then observed by laser confocal microscope (OLYMPUS FV1000-IX81, Olympus, Japan). For flow cytometry, CT26 cells were seeded in 12-well plates (2× 10 5 cells/well) and cultured for 12 h. After treatment with The absorbance of the well was tested by a Microplate reader (SpectraMax) at 570 nm (peak absorbance) and subtracted at 650 nm (background absorbance). Cell viability was expressed as the ratio of the absorbance of the test and control wells. In Vivo FL Imaging and Biodistribution Analysis: CT26 cells (1 × 10 6 ) were subcutaneously injected into the right hip of female BALB/c mice. When tumor volumes reached about 100 mm 3 , mice were injected with NP-Cy5.5 iv. After injection, FL signals were recorded using the IVIS spectrum imaging system (Spectrum CT, PerkinElmer) at 1, 3, 9, 12, 24, 36 h, respectively. For the biodistribution study, mice were sacrificed after 36 h after injection and tumors and normal organs were harvested and imaged.

Cell apoptosis of S-NP-CPT in
Pharmacokinetic study of S-NP-CPT: CT26 cells (1 × 10 6 ) were subcutaneously injected into the right hip of female BALB/c mice. When tumor volumes reached about 100 mm 3 , mice were injected with NP-Cy5.5 iv. After injection, at each required time point (5 min, 0.5, 1, 3, 6, 9, 12, 24 h) 10 μL of plasma was collected from the tail vein and then the concentration of Cy5.5 in the plasma was measured by fluorescence.
Subsequently, the tumors were sectioned and observed by CLSM. Statistical analysis：The level of significance in all statistical analyses was set at P < 0.05. Data were analyzed using a two-sided Student's t-test when two groups were being compared. One-way or two-way analysis of variance (ANOVA) and Tukey posthoc tests were used when more than two groups were compared (multiple comparisons) using Prism 8.0 (GraphPad Software).